2021 Fiscal Year Annual Research Report
New cryo-electron microscopy method for elucidation of G-protein coupled receptor supercomplexes
| Project/Area Number |
21F20764
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| Research Institution | The University of Tokyo |
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Principal Investigator |
Danev Radostin 東京大学, 大学院医学系研究科(医学部), 教授 (50415931)
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| Co-Investigator(Kenkyū-buntansha) |
EISENSTEIN FABIAN 東京大学, 医学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2021-04-28 – 2023-03-31
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| Keywords | cryo-electron microscopy / single particle analysis / GPCR / cryo-electron tomography |
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| Outline of Annual Research Achievements |
The first year of the research was focused on the establishment of experimental workflows and the optimization of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) parameters. Single particle G protein-coupled receptor (GPCR) samples were investigated by cryo-EM and the effect of several experimental factors were quantitatively evaluated. The results indicated that the Volta phase plate (VPP) had a strong negative impact, while zero-loss energy filtering and higher electron exposure had a positive influence on the resolution of reconstructed 3D maps. The use of gold foil support grids also had a strong positive effect on resolution by improving the uniformity and reproducibility of good quality thin ice and by reducing the beam-induced motion of the specimen. These results allowed us to establish an optimal single-particle experimental workflow for GPCRs.
Following the cryo-EM refinement, the research transitioned to establishment of the in situ cryo-ET workflow for GPCRs. The sample freezing conditions for adherent cellar culture samples were optimized. Frozen samples were transferred to a cryo-focused ion beam (cryo-FIB) instrument for thinning. The cryo-FIB milling process was automated using the AutoTEM software, which allowed the preparation of more than 10 lamellas per day. Milled samples were transferred to a cryo-electron microscope for cryo-ET data collection and the quality of the samples was confirmed. An innovative deep-learning based approach for cryo-ET data acquisition was developed and tested with overall very promising results.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
The results from the first year of research exceeded our expectations. Our quantitative evaluation and optimization of cryo-EM GPCR experimental parameters was received with a lot of excitement by the cryo-EM and GPCR structural communities. It allowed us to fine-tune our experiments and achieve unprecedented results in terms of particle size and resolution. Furthermore, our initial trials in cellular sample freezing, cryo-FIB milling, and cryo-tomography, showed that all steps of the workflow have good performance and consistency and that future performance gains will be possible, towards GPCRs visualization in their native cellular environment.
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| Strategy for Future Research Activity |
The next steps in the project will focus on in situ cryo-ET investigations of GPCRs and other membrane proteins. The sample freezing, cryo-FIB milling, and cryo-ET workflows will be optimized individually and with careful attention to detail.
On the specimen side, we will test several cryo-protectant buffers with the aim of minimizing the nano-crystallization that is sometimes observed inside the frozen cellular volumes. It generates crystalline reflection spots and disturbs cellular structures.
In the cryo-FIB workflow, we plan to improve the automated milling protocol for achieving a higher success rate and better-quality lamellas.
For cryo-ET, we are planning to improve the experimental throughput and the quality of the data through innovative data acquisition approaches.
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